Fig. 1. Morphological changes in molar spatial patterning in mouse after maternal transfer of
5E1. (A-J) Five types of molar patterns obtained in maxilla (Mx) and mandible (Mn): Type-I, wildtype-like with first (M1), second (M2) and third (M3) molar; Type-II, M1-M2 fusion; Type-III, M1M2 fusion with extra lingual cusps (arrows); Type-IV, M2-M3 fusion; Type-V, M1-M2-M3 fusion.
(K-N) Three-dimensional micro-computed-tomography images showing buccal aspect of type-I and
type-II patterns at embryonic day (E) 14 after injection of phosphate-buffered saline (E14-PBS) and
after 5E1 injection (E14-5E1) in maxilla and mandible. (O-T) Dental lamina (arrow) is evident in
the frontal section of M1 in both E14-PBS and E14-5E1 at one day after injection (O,R). After two
days, dental lamina and lingual epithelial bud (arrowhead) are observed only in an E14-PBS
(compare P with S), and M1-M2 separation (red arrow) is evident in E14-PBS (Q). M1-M2 fusion
is evident in E14-5E1 (T). (U,V) Three-dimensional images of dental epithelium from bottom view
show the larger buccolingual diameter of M2 (between arrows) in E14-5E1 (V) than in E14-PBS
(U) after one day. The boundary (red arrowheads) between M1 and M2 is clearly shown in E14PBS after two days both from bottom and buccal view (U), whereas M1-M2 fusion is evident in
E14-5E1 (V). Scale bars: 500 μm.
Fig. 2.
Morphological changes in mouse molars after 5E1 treatment in vivo. (A,B) Supernumerary
incisors (red arrow) develop in embryonic day (E) 14 mice treated with 5E1 (B, E14-5E1; 2/82 in
mandibular quadrants) but not in control mice treated with PBS (A, E14-PBS). (C-E)
Supernumerary molars (arrowheads) develop in E12-5E1 [1/40 in the maxillary quadrants (C), 1/32
in the mandibular quadrants], E14-5E1 (4/78 in the maxillary quadrants, D) and E16-5E1 (1/54 in
the mandibular quadrants, E). (F,G) Transparent enamel (arrows) is seen covering the dentine in
sagittal sections of fused molars in Mx and Mn of E14-5E1. (H-K) Three-dimensional images from
occlusal view show pattern of molars in 4-week-old E14-PBS and E14-5E1. E14-PBS mice have
three molars in Mx (H) and Mn (J). M1-M2 fusion is observed in Mx (I) and Mn (K) of E14-5E1.
Extra cusps are evident on lingual side of a fused molar (red arrowheads in K). Scale bars: 1 mm.
Mx, maxilla; Mn, mandible.
Fig. 3.
Transcriptional changes in molars after maternal transfer of 5E1 into mouse embryos at E14.
RT-qPCR analysis shows up- or downregulation of Ptch1, Hhip, Gli1, Sostdc1, Sp5, Fgf4 and
Krt73. The amount of each of the RT-qPCR products was normalized using β-2-microglobulin
(B2m) as an internal control. Student's t-test was performed for statistical analysis with level of
statistical significance set at P<0.05. Error bars indicate s.d. on the normalized ratio.
Fig. 4.
Alterations in gene expression pattern in 5E1-treated mouse molars. (A-D) Whereas the pattern
of Shh expression is the same in E14-PBS and E14-5E1 mice after one day in vivo (A,B), Shh
expression in M2 (black arrowhead) is evident only in E14-5E1 in vitro, showing accelerated M2
development (compare D with C). (E-H) Ptch1 is strongly expressed in E14-PBS in vivo (E) and in
vitro (G), but disappeared in E14-5E1 (F,H). (I-L) Sostdc1-expressing areas are markedly reduced
in E14-5E1 (J,L) compared with E14-PBS (I,K). (M-P) Sp5 expression in M1 and M2 is separated
in E14-PBS in vivo (red arrowhead in M). Sp5 is observed in M1 of E14-PBS after one day (+1D)
culture in vitro (O) and expressed in M1 and M2 separately after two days (+2D) (red arrowhead in
O′), but connected in E14-5E1 in vivo and from one day in culture (white arrowheads in N and P).
(Q,R) Gli3 expression pattern of E14-PBS is the same as that of E14-5E1 after one day in vitro.
(S,T) Lef1 expression is observed in M1 after one day in vitro in both E14-PBS and E14-5E1, but
its domain of expression in M2 (red arrows) is larger in E14-5E1 (T) than in E14-PBS (S). (U,V)
krt73 is expressed in a line in E14-PBS (U), but is widely expressed throughout M1 and M2 in E145E1 (V). (W,X) Fgf4 expression in M1 (black arrow) is evident in both E14-PBS (W) and E14-5E1
(X), but its expression in M2 (red arrow) is only present in E14-5E1 (X). Scale bar: 200 μm
Fig. 5.
Effects of Shh on Sostdc1 expression in mouse. (A-D) Apart from endogenous Ptch1 (red arrows
in A and B), exogenous Ptch1 is expressed around the Shh protein bead in both dental epithelium
and mesenchyme (black arrows in B,D), but not around PBS beads (A,C). (E-H) Sostdc1 is not
expressed around the control PBS beads (E,G). Sostdc1 is induced by exogenous Shh protein in
mesenchyme (arrow in H), but not in epithelium (F). (I-L) Sostdc1 is expressed in dental epithelium
and mesenchyme around wild-type tooth germs at E14 but is not induced in either epithelium or
mesenchyme around beads soaked in Shh protein. Occlusal views are shown in I and K. Frontal
section is shown in J and L at the level of the dashed lines in I and K, respectively. Yellow dashed
lines indicate the boundary of the dental epithelium and mesenchyme. Scale bars: 200 μm.
Fig. 6.
Wnt-Shh-Sostdc1 negative feedback loop for the regulation of the tooth spatial patterning.
(A,B) Frontal section of M1 at E14 shows that Lef1 is expressed in the enamel knot (inside the red
dashed circles) and dental mesenchyme surrounding the enamel knot, whereas Sostdc1 is expressed
mainly outside the Lef1-expressing area. (C) Schematic of Wnt-Shh-Sostdc1 negative feedback
loop in M1 at E14 from frontal view. Wnt signals in the enamel knot induce Shh in the enamel knot
and Wnt moves laterally to induce Lef1 and Sp5. Secreted Shh in the enamel knot also moves
laterally to induce Sostdc1. The Sostdc1-expressing area (Sostdc1 area) is non-overlapping with the
Lef1-expressing area (Lef1 area). (D) Wild-type mouse has three molars, each of which has an
activation zone (Lef1 area) and an inhibition zone (Sostdc1 area). Loss of the inhibition zone in
K14-Cre;Shhflox/flox, K14-Cre;Smoflox/flox, Sostdc1−/−, Lrp4−/− and 5E1-transferred mice enhances
molar fusion. Sustained Wnt/β-catenin signals in K14-Cre;Ctnnb1(Ex3)fl/+ and K14-Cre;APCcko/cko
induce multiple activation and inhibition zones to form multiple molars. (E) Schematic of the
proposed reaction-diffusion model showing interactions between activator (A), mediator (M) and
inhibitor (I). (F) Simulated patterns of activator, mediator and inhibitor show three molars in wildtype mice. The black dotted lines in A-C indicate the boundary of the dental epithelium and
mesenchyme. Scale bar: 100 μm.